Diversity and evolutionary patterns of bacterial gut associates of corbiculate bees

The animal gut is a habitat for diverse communities of microorganisms (microbiota). Honeybees and bumblebees have recently been shown to harbour a distinct and species poor microbiota, which may confer protection against parasites. Here, we investigate diversity, host specificity and transmission mode of two of the most common, yet poorly known, gut bacteria of honeybees and bumblebees: Snodgrassella alvi (Betaproteobacteria) and Gilliamella apicola (Gammaproteobacteria). We analysed 16S rRNA gene sequences of these bacteria from diverse bee host species across most of the honeybee and bumblebee phylogenetic diversity from North America, Europe and Asia. These focal bacteria were present in 92% of bumblebee species and all honeybee species but were found to be absent in the two related corbiculate bee tribes, the stingless bees (Meliponini) and orchid bees (Euglossini). Both Snodgrassella alvi and Gilliamella apicola phylogenies show significant topological congruence with the phylogeny of their bee hosts, albeit with a considerable degree of putative host switches. Furthermore, we found that phylogenetic distances between Gilliamella apicola samples correlated with the geographical distance between sampling locations. This tentatively suggests that the environmental transmission rate, as set by geographical distance, affects the distribution of G. apicola infections. We show experimentally that both bacterial taxa can be vertically transmitted from the mother colony to daughter queens, and social contact with nest mates after emergence from the pupa greatly facilitates this transmission. Therefore, sociality may play an important role in vertical transmission and opens up the potential for co‐evolution or at least a close association of gut bacteria with their hosts.     

Diversity and functional analysis of Chinese bumblebee gut microbiota reveal the metabolic niche and antibiotic resistance variation of Gilliamella

Bumblebees play an important role in maintaining the balance of natural and agricultural ecosystems,and the characteristic gut microbiota of bumblebees exhibit significant mutualistic functions.China has the highest diversity of bumblebees;however,gut microbiota of Chinese bumblebees have mostly been investigated through cultureindependent studies.Here,we analyzed the gut communities of bumblebees from Sichuan,Yunnan,and Shaanxi provinces in China through 16S ribosomal RNA amplicon sequencing and bacterial isolation.It revealed that the bumblebees examined in this study harbored two gut enterotypes as previously reported:one is dominated by Gilliamella and Snodgrassella,and the other is distinguished by prevalent environmental species.The gut compositions obviously varied among different individual bees.We then isolated 325 bacterial strains and the comparative genomic analysis of Gillianiella strains revealed that galactose and pectin digestion pathways were conserved in strains from bumblebees,while genes for the utilization of arabinose,mannose,xylose,and rhamnose were mostly lost.Only two strains from the Chinese bumblebees possess the multidrug-resistant gene emrB,which is phylogenetically closely related to that from the symbionts of soil entomopathogenic nematode.In contrast,tetracycline-resistant genes were uniquely present in three strains from the USA.Our results illustrate the prevalence of strain-level variations in the metabolic potentials and the distributions of antibiotic-resistant genes in Chinese bumblebee gut bacteria.     

Thermal niches of specialized gut symbionts: the case of social bees

Responses to climate change are particularly complicated in species that engage in symbioses, as the niche of one partner may be modified by that of the other. We explored thermal traits in gut symbionts of honeybees and bumblebees, which are vulnerable to rising temperatures. In vitro assays of symbiont strains isolated from 16 host species revealed variation in thermal niches. Strains from bumblebees tended to be less heat-tolerant than those from honeybees, possibly due to bumblebees maintaining cooler nests or inhabiting cooler climates. Overall, however, bee symbionts grew at temperatures up to 44°C and withstood temperatures up to 52°C, at or above the upper thermal limits of their hosts. While heat-tolerant, most strains of the symbiont Snodgrassella grew relatively slowly below 35°C, perhaps because of adaptation to the elevated body temperatures that bees maintain through thermoregulation. In a gnotobiotic bumblebee experiment, Snodgrassella was unable to consistently colonize bees reared at 29°C under conditions that limit thermoregulation. Thus, host thermoregulatory behaviour appears important in creating a warm microenvironment for symbiont establishment. Bee–microbiome–temperature interactions could affect host health and pollination services, and inform research on the thermal biology of other specialized gut symbionts.     

16S rRNA Amplicon Sequencing Demonstrates that Indoor-Reared Bumblebees (Bombus terrestris) Harbor a Core Subset of Bacteria Normally Associated with the Wild Host

A MiSeq multiplexed 16S rRNA amplicon sequencing of the gut microbiota of wild and indoor-reared Bombus terrestris (bumblebees) confirmed the presence of a core set of bacteria, which consisted of Neisseriaceae (Snodgrassella), Orbaceae (Gilliamella), Lactobacillaceae (Lactobacillus), and Bifidobacteriaceae (Bifidobacterium). In wild B. terrestris we detected several non-core bacteria having a more variable prevalence. Although Enterobacteriaceae are unreported by non next-generation sequencing studies, it can become a dominant gut resident. Furthermore the presence of some non-core lactobacilli were associated with the relative abundance of bifidobacteria. This association was not observed in indoor-reared bumblebees lacking the non-core bacteria, but having a more standardized microbiota compared to their wild counterparts. The impact of the bottleneck microbiota of indoor-reared bumblebees when they are used in the field for pollination purpose is discussed.     

The Value of Uncropped Field Margins For Foraging Bumblebees

The intensification of agriculture has led to declines in species diversity and abundance within groups of certain flora and fauna. Bumblebees (Bombus spp.) are one group where a decline has been documented, and it is thought to be attributable to a decrease in forage resources and potential nest sites. As bumblebees play an important role in the pollination of many entomophilous crops, this decline could impact on agricultural productivity. We examined the role of naturally regenerated field margins in providing forage plants on land where nectar resources are otherwise impoverished. The following question was addressed – Are naturally regenerated unsprayed field margins more attractive to foraging bumblebees and honeybees than cropped field margins managed as conservation headlands? Significantly more bees visited naturally regenerated field margins than cropped field margins. Honeybees (Apis mellifera), Bombus terrestris, and Bombus lapidarius were the most commonly observed bee species. Different wildflower species within the naturally regenerated margins varied greatly in relative number of visits received, and bumblebee species were found to prefer different flower species to honeybees. The potential role that naturally regenerated field margins could play in the conservation of bumblebee species, and the implications for other species of flora and fauna, are discussed.     

Evidence for competition between honeybees and bumblebees; effects on bumblebee worker size

Numerous studies suggest that honeybees may compete with native pollinators where introduced as non-native insects. Here we examine evidence for competition between honeybees and four bumblebee species in Scotland, a region that may be within the natural range of honeybees, but where domestication greatly increases the honeybee population. We examined mean thorax widths (a reliable measure of body size) of workers of Bombus pascuorum, B. lucorum, B. lapidarius and B. terrestris at sites with and without honeybees. Workers of all four species were significantly smaller in areas with honeybees. We suggest that reduced worker size is likely to have implications for bumblebee colony success. These results imply that, for conservation purposes, some restrictions should be considered with regard to placing honeybee hives in or near areas where populations of rare bumblebee species persist.     

Do exotic bumblebees and honeybees compete with native flower-visiting insects in Tasmania?

Honeybees, Apis mellifera, have been introduced by man throughout the globe. More recently, other bee species including various bumblebees (Bombus spp.) have been introduced to several new regions. Here we examine the impacts of honeybees and the bumblebee, Bombus terrestris, on native flower-visiting insects in Tasmania. To assess whether native insects have lower abundance or are excluded in areas that have been colonised by exotic bees, we quantified the abundance, diversity and floral preferences of flower-visiting insects at sites where bumblebees and honeybees were present, and compared them to sites where they were absent. This was achieved by hand searches at 67 sites, and by deploying sticky traps at 122 sites. Honeybees were by far the most abundant bee species overall, and dominated the bee fauna at most sites. There was considerable niche overlap between honeybees, bumblebees and native bees in terms of the flowers that they visited. Sites where bumblebees were established had similar species richness, diversity and abundance of native flower-visiting insects compared to sites where bumblebees were absent. In contrast, native bees were more than three times more abundant at the few sites where honeybees were absent, compared to those where they were present. Our results are suggestive of competition between honeybees and native bees, but exclusion experiments are needed to provide a definitive test.     

Abundance,body size,and morphology of bumblebees in an area where an exotic species, <Emphasis Type="Italic">Bombus terrestris</Emphasis>, has colonized in Japan

An exotic bumblebee species, Bombus terrestris, has colonized in Japan and becomes dominant in some local communities. We examined the effects of land use and bumblebee abundance on the number and body size of bumblebees collected using window traps in a lowland area in the southern Ishikari district, Hokkaido. In 2004, we collected 922 bumblebees of six species using 70 traps at 17 sites. A statistical model fitted to the data demonstrated that dispersion from commercial B. terrestris colonies used in greenhouses positively affected the number of B. terrestris caught by each trap. This exotic species was abundant in sites where paddy fields were prevalent, but three native species, B. hypocrita, B. ardens, and B. diversus, were abundant in sites where farms and woodlands were widespread. The local abundance of B. terrestris was not associated negatively with the number and body size of native bumblebees. Thus, we did not find any competitive interactions between exotic and native bumblebees although habitat conditions seem to be common determinants of the bumblebee populations. A morphological analysis showed that B. terrestris had intermediate tongue length between B. hypocrita and B. ardens.     

Visual detection of diminutive floral guides in the bumblebee <Emphasis Type="Italic">Bombus terrestris</Emphasis> and in the honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis>

Many flowers display colour patterns comprising a large peripheral colour area that serves to attract flower visitors from some distance, and a small central, contrastingly coloured area made up by stamens or floral guides. In this study, we scaled down the size of floral guides to detect the minimal size bumblebees (Bombus terrestris) and honeybees (Apis mellifera) require for guidance. We analyzed the approach and the precise contact of the antennal tips with the floral guide of artificial flowers which precedes landing and inspection. Both bumblebees and honeybees were able to make antennal contact with circular floral guides which were 2 mm in diameter; bumblebees performed better than honeybees and antennated also at floral guides smaller than 2 mm. In discrimination experiments with bumblebees, a minimum floral guide size of 2 mm was required for discrimination between artificial flowers with and without floral guides. With increasing experience bumblebees targeted close to the site of reward instead of making antennal contact with the floral guide, whereas honeybees did not alter their initial behaviour with growing experience. Bumblebees and honeybees spontaneously target diminutive floral guides to achieve physical contact with flowers by means of their antennae which helps them to inspect flowers.     

Induction of colony initiation by Japanese native bumble bees using cocoons of the exotic bumblebee Bombus terrestris

The colony initiation rates of Bombus hypocrita (a native Japanese bumblebee) and Bombus terrestris (a European species) foundresses were compared after 4 weeks of exposure to B. terrestris cocoons. The B. terrestris cocoons, when replaced weekly, were effective for inducing oviposition by foundresses of both species. There were no significant differences in the colony initiation rates of B. terrestris and B. hypocrita, either with the control treatment or with the cocoons. The cocoon method was also tested for five species and two subspecies of native Japanese bumblebees. The colony initiation rate was higher for foundresses of the subgenus Bombus s. str. than for foundresses of the subgenera Pyrobombus, Diversobombus, and Thoracobombus. When replaced weekly, the cocoons of B. terrestris are effective inducers of colony foundation in three Japanese native species, namely B. ignitus, B. hypocrita hypocrita, and B. h. sapporoensis.     

Targeted agri‐environment schemes significantly improve the population size of common farmland bumblebee species

Changes in agricultural practice across Europe and North America have been associated with range contractions and local extinction of bumblebees (Bombus spp.). A number of agri‐environment schemes have been implemented to halt and reverse these declines, predominantly revolving around the provision of additional forage plants. Although it has been demonstrated that these schemes can attract substantial numbers of foraging bumblebees, it remains unclear to what extent they actually increase bumblebee populations. We used standardized transect walks and molecular techniques to compare the size of bumblebee populations between Higher Level Stewardship (HLS) farms implementing pollinator‐friendly schemes and Entry Level Stewardship (ELS) control farms. Bumblebee abundance on the transect walks was significantly higher on HLS farms than ELS farms. Molecular analysis suggested maximum foraging ranges of 566 m for Bombus hortorum, 714 m for B. lapidarius, 363 m for B. pascuorum and 799 m for B. terrestris. Substantial differences in maximum foraging range were found within bumblebee species between farm types. Accounting for foraging range differences, B. hortorum (47 vs 13 nests/km²) and B. lapidarius (45 vs 22 nests/km²) were found to nest at significantly greater densities on HLS farms than ELS farms. There were no significant differences between farm type for B. terrestris (88 vs 38 nests/km²) and B. pascuorum (32 vs 39 nests/km²). Across all bumblebee species, HLS management had a significantly positive effect on bumblebee nest density. These results show that targeted agri‐environment schemes that increase the availability of suitable forage can significantly increase the size of wild bumblebee populations.     

Foraging interactions between native and exotic bumblebees: enclosure experiments using native flowering plants

To assess the impact of Bombus terrestris invasion on the foraging efficiency of native Japanese bumblebees, consumption and acquisition of floral resources during foraging on flowers of native Japanese plant species were investigated using enclosures with three treatments: one with only B. terrestris (exotic), one with both B. terrestris and native Japanese bumblebee species (mixed), and one with only Japanese species (native), but with the bumblebee density held constant. Changes in the body mass of queens and the nest mass of colonies for two days did not significantly differ among four combinations of the species and treatment, B. terrestris in the exotic and mixed treatments and Japanese species in the mixed and native treatments. Thus, it is not clear that B. terrestris has higher foraging efficiency than native species and that B. terrestris individuals more negatively affect the foraging efficiency of native species than individuals of the native species themselves. The nectar standing crop of Cirsium kamtschaticum was smaller in the exotic treatment than in the mixed and native treatments. However, this may have been an artifact of differences in the numbers of flowers in the various treatments. T. Nagamitsu and T. Kenta contributed equally to this work     

Potential host shift of the small hive beetle (Aethina tumida) to bumblebee colonies (Bombus impatiens)

Here we explored the potential for host shift from honeybee, Apis mellifera, colonies to bumblebee, Bombus impatiens, colonies by the small hive beetle, a nest parasite/scavenger native to sub-Saharan Africa. We investigated small hive beetle host choice, bumblebee colony defence as well as individual defensive behaviour of honeybee and bumblebee workers. Our findings show that in its new range in North America, bumblebees are potential alternate hosts for the small hive beetle. We found that small hive beetles do invade bumblebee colonies and readily oviposit there. Honeybee colonies are not preferred over bumblebee colonies. But even though bumblebees lack a co-evolutionary history with the small hive beetle, they are able to defend their colonies against this nest intruder by removal of beetle eggs and larvae and stinging of the latter. Hence, the observed behavioural mechanisms must be part of a generalistic defence system suitable for defence against multiple attackers. Nevertheless, there are quantitative (worker force) and qualitative differences (hygienic behaviour) between A. mellifera and B. impatiens. Received 16 July 2007; revised 16 January 2008; accepted 17 January 2008.     

Bombus terrestris in a mass‐flowering pollinator‐dependent crop: A mutualistic relationship?

Bumblebees (Bombus spp.) rely on an abundant and diverse selection of floral resources to meet their nutritional requirements. In farmed landscapes, mass‐flowering crops can provide an important forage resource for bumblebees, with increased visitation from bumblebees into mass‐flowering crops having an additional benefit to growers who require pollination services. This study explores the mutualistic relationship between Bombus terrestris L. (buff‐tailed bumblebee), a common species in European farmland, and the mass‐flowering crop courgette (Cucurbita pepo L.) to see how effective B. terrestris is at pollinating courgette and in return how courgette may affect B. terrestris colony dynamics. By combining empirical data on nectar and pollen availability with model simulations using the novel bumblebee model Bumble‐BEEHAVE, we were able to quantify and simulate for the first time, the importance of courgette as a mass‐flowering forage resource for bumblebees. Courgette provides vast quantities of nectar to ensure a high visitation rate, which combined with abundant pollen grains, enables B. terrestris to have a high pollination potential. While B. terrestris showed a strong fidelity to courgette flowers for nectar, courgette pollen was not found in any pollen loads from returning foragers. Nonetheless, model simulations showed that early season courgette (nectar) increased the number of hibernating queens, colonies, and adult workers in the modeled landscapes. Synthesis and applications. Courgette has the potential to improve bumblebee population dynamics; however, the lack of evidence of the bees collecting courgette pollen in this study suggests that bees can only benefit from this transient nectar source if alternative floral resources, particularly pollen, are also available to fulfill bees’ nutritional requirements in space and time. Therefore, providing additional forage resources could simultaneously improve pollination services and bumblebee populations.     

Collective control of nest climate parameters in bumblebee colonies

We examined two aspects of the social control of nest climate in bumblebee colonies: which parameters of nest climate bumblebees actively down-regulate by fanning and the dynamics of the colony response as colony size increased. Colonies of Bombus terrestris were exposed to an increase in carbon dioxide, temperature or relative humidity. We performed 70 temperature trials (six colonies), 58 CO₂ trials (four colonies) and four humidity trials (two colonies). An increase in CO₂ concentration and temperature elicited a fanning response whereas an increase in relative humidity did not. This is the first report of fanning in bumblebee colonies to control respiratory gases. The number of fanning bees increased with stimulus intensity. The colony response to a CO₂ concentration of 3.2% was comparable to the colony response to a temperature of 30°C. A marked fanning response occurred at 1.6% CO₂, a concentration never exceeded in a large field nest during a pilot measurement of 10 days. We investigated the colony response over a wide range of colony sizes (between 10 and 119 workers). The proportion of the total workforce invested by colonies in nest ventilation did not change significantly; thus, the number of fanning workers increased with colony size. Furthermore, as colony size increased, the dynamics of the colony response changed: colonies responded faster to perturbations of their environment when they were large (60 or more individuals) than when they were small. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Displacement of Japanese native bumblebees by the recently introduced <Emphasis Type="Italic">Bombus terrestris</Emphasis> (L.) (Hymenoptera: Apidae)

The introduced Bombus terrestris has recently been naturalized in Japan and become dominant in some local communities. We investigated potential niche overlaps between introduced and native bumblebees in terms of morphological characteristics, seasonal flight activity, foraging and nesting habitat use, and plant species visited. There were considerable niche overlaps in flower resource use between B. terrestris and B. hypocrita sapporoensis/B. pseudobaicalensis. Bombus terrestris also potentially competes for nest sites with B. hypocrita sapporoensis. During 3-year monitoring, B. pseudobaicalensis showed no noticeable change, but B. hypocrita sapporoensis decreased while B. terrestris increased. Abundant flower resources provided by exotic plants may buffer native bumblebees from competition for food with introduced species. By contrast, the number of nest usurpers found in B. terrestris nests increased between 2003 and 2005, indicating that availability of nest sites was limiting and queens strongly competed for nest sites. Our findings suggest that competition for nest sites rather than flower resources is the major ecological mechanism for displacement of native bees. The large reduction of B. hypocrita sapporoensis queen indicates that B. terrestris may cause local extinction of native bumblebees. Control of established B. terrestris populations and prevention of further range expansion are urgently needed.     

Construction and characterization of a BAC-library for a key pollinator, the bumblebee Bombus terrestris L.

The primitively social bumblebee Bombus terrestris is an ecological model species as well as an important agricultural pollinator. As part of the ongoing development of genomic resources for this model organism, we have constructed a publicly available bacterial artificial chromosome (BAC) library from males of a field-derived colony. We have shown that this library has a high coverage, which allows any particular sequence to be retrieved from at least one clone with a probability of 99.7%. We have further demonstrated the library’s usefulness by successfully screening it with probes derived both from previously described B. terrestris genes and candidate genes from another bumblebee species and the honeybee. This library will facilitate genomic studies in B. terrestris and will allow for novel comparative studies in the social Hymenoptera. Received 12 March 2008; revised 13 September 2008; accepted 17 September 2008. R. Schmid-Hempel: contact for data repository of this BAC library     

Microbial Communities of Three Sympatric Australian Stingless Bee Species

Bacterial symbionts of insects have received increasing attention due to their prominent role in nutrient acquisition and defense. In social bees, symbiotic bacteria can maintain colony homeostasis and fitness, and the loss or alteration of the bacterial community may be associated with the ongoing bee decline observed worldwide. However, analyses of microbiota associated with bees have been largely confined to the social honeybees (Apis mellifera) and bumblebees (Bombus spec.), revealing – among other taxa – host-specific lactic acid bacteria (LAB, genus Lactobacillus) that are not found in solitary bees. Here, we characterized the microbiota of three Australian stingless bee species (Apidae: Meliponini) of two phylogenetically distant genera (Tetragonula and Austroplebeia). Besides common plant bacteria, we find LAB in all three species, showing that LAB are shared by honeybees, bumblebees and stingless bees across geographical regions. However, while LAB of the honeybee-associated Firm4–5 clusters were present in Tetragonula, they were lacking in Austroplebeia. Instead, we found a novel clade of likely host-specific LAB in all three Australian stingless bee species which forms a sister clade to a large cluster of Halictidae-associated lactobacilli. Our findings indicate both a phylogenetic and geographical signal of host-specific LAB in stingless bees and highlight stingless bees as an interesting group to investigate the evolutionary history of the bee-LAB association.     

Dynamics of gut microbiome upon pollination in bumblebee (Bombus terrestris)

Bumblebees are crucial buzz pollinators of poricidal plants and commercial crops. The population of these crucial pollinators is globally declining. The need to focus on factors contributing to bee health such as gut microbiota is imperative. We evaluated the effect of source on gut microbiota composition in adult worker Bombus terrestris from two environments using multiplexed 16S rRNA amplicon sequencing on the Illumina MiSeq platform. Indoor reared adult worker bees were segregated into non-pollination (NP) and pollination (P) groups. The NP group bumblebees were raised and kept in the insectary for the entire experiment. P group bumblebees worked in a tomato-planted greenhouse for 15 days as pollinators. Our results show that members of Proteobacteria and Firmicutes were significantly different between the two groups. Bifidobacterium bombi (Actinobacteriota) and Candidatus_Schmidhempelia (Proteobacteria) were found to be important gut colonizer exclusive to NP and P group bees, respectively. DESeq2 analysis showed differentially abundant OTUs belonging to Lactobacillus spp. in each group. But for the differentially abundant detected OTUs, resolution till genus level was obtained which impairs our knowledge of the species associated with each group. Our data show that the gut microbiota between the groups differed when indoor- reared adult worker bees were exposed to different environments. The distinct gut microbiota between the two groups may also have been influenced by the different diets fed to bees upon segregation. Further studies can give important insights into the role of gut microbiota on bee health when indoor reared bees are employed for pollination.